Evaluating the Effect of an Experimental Sesame Gel on Human Enamel: Atomic Force Microscopy Study

Statement of the Problem:
 Every effort for increasing the calcium concentration in the saliva would be beneficial for prevention of dental caries. Regarding this issue, the natural products could be considered safer and more cost effective. Sesame is rich in calcium but the data about the effect of sesame on enamel roughness is inadequate. 
Purpose:
 This study aimed to assess the effect of an experimental sesame gel on the surface roughness of human enamel by using atomic force microscopy (AFM). 
Materials and Method:
 In the current experimental study, fifteen enamel slices with 1mm thickness were prepared. They were polished and acid etched to produce a substantial rough surface prior to the first AFM analysis. The enamel blocks were randomly divided into three groups treated with distilled water, fluoride gel, and an experimental prepared sesame gel correspondingly. The treating agent was applied for 3minutes at 0, 8, 24, and 48h intervals and washed by distilled water after each cycles. Ultimately, the final AFM micrographs were prepared. The statistical analysis was performed using paired t-test, one-way ANOVA, and Tukey Post Hoc tests (α=0.05). 
Results:
 Statistical analysis revealed that the surface roughness was significantly reduced in both sesame and fluoride groups (p= 0.017 and 0.018, respectively) while the control group (distilled water) were not noticeably changed (p= 0.12). The control group had statistically significant difference with both the sesame and the fluoride groups (p= 0.007 and 0.007, respectively) while the there was no significance difference between sesame and fluoride groups (p= 0.997). 
Conclusion:
 Following demineralization by acid etched process, the sesame gel significantly reduced surface roughness of enamel and its effect was similar to fluoride gel.


Introduction
Dental caries is a major public health problem and the most common infectious disease among human population [1]. Since most dental treatments are painful and costly, patients have become more interested in preventing tooth decay rather than seeking treatment [2].
Carious lesions are the result of demineralization of the tooth microstructure due to the acid produced by cariogenic bacteria, which subsequently lead to cavity production in tooth structure [3]. Therefore, remineralizing agents that expose the tooth surface to mineral-rich saliva [4] might slow down or even prevent the decay process [3]. Despite the rich history of the benefits of fluoride in tooth care and its robust status as the standard low-cost therapeutic agent in dental clinics [2], it has some shortcomings such as toxicity [5][6][7][8]. Consequently, recent studies are conducted to offer some innovative and very effective preventive agents such as bioactive glasses, amorphous calcium phosphate, and so on. Although these agents showed remarkable remineralization potentials, their high cost and the need for modern mobile technologies have limited their use in dental clinics [3,6,[8][9][10].
In recent years, dental researchers have been encouraged to investigate the effects of natural products on dental caries [11]; these products generally show minimal or no side effects and they are economic [12].
The research shows that some foods contain anti-decay agents such as calcium, phosphate, and casein. Hence, these foods might potentially be able to contribute to remineralization process and reduce the occurrence of caries [13]. Moreover, green leafy vegetables, seeds, and legumes are rich sources of calcium [14], among which, sesame is most remarkable and famous one [15].
Besides that, in traditional medicine, oil therapy has been widely recommended to strengthen the teeth, jaws, gums, and prevent teeth decay [16]. Sesame oil has been documented as one of the most frequent prescribed oils in traditional medicine for this purpose [16]. Previous studies have provided evidence that oil therapy with sesame oil could be used for prevention of tooth decay [17][18][19]. It is also reported that the sesame oil could noticeably reduce the plaque formation and consequently inhibit the plaque induced gingivitis [4,18]. In addition, the antibacterial potential of sesame oil has been broadly documented against the cariogenic bacteria particularly Streptococcus mutans (S.mutans) [19][20]. Therefore, the sesame seed could be considered as an effective substance in preventive dentistry [4,[18][19][20]. Moreover, numerous anti-caries agents have remineralization capability regarding their calcium or other mineral contents. Current studies have shown that sesame is one of the richest herbal sources of calcium [14,17].
However, the available data regarding the effect of sesame on surface enamel is quite sparse. In an in vitro study, it has been shown that this plant could increase the microhardness of human enamel though the effect of sesame to reduce the surface roughness especially after acid incorporation has not been investigated [21]. The effect of sesame on remineralization process is still disputed. Therefore, this study aimed to investigate the effect of an experimental sesame gel on the surface roughness of human enamel by employing atomic force microscopy (AFM).

Materials and Method Preparation of sesame extract gel
In order to prepare the extract, 200 gr of black sesame seed, purchased locally, were immersed in boiled water for 48 h. Consequently, they were filtrated, the solvent was evaporated, and the residues were weighed that was roughly 20gr. Finally, it was admixed with 80 gr of Carbomer 934 to produce an applicable 20% gel.

Preparation of the teeth
The Research Ethics Committee of Kerman University of Medical Science (2/2017) approved the study. Eight healthy human premolars, extracted for orthodontic reasons, were selected for this study. The teeth were stored in tap water while the water was replaced once a week. At the beginning of the study, the teeth were disinfected by immersion in 5.25% sodium hypochlorite solution for one hour, and then stored in distilled water.
In order to discard the unacceptable samples, the teeth were cleaned with a low-speed handpiece using brushes and slurry of pumice. The teeth were examined by naked eye and dental loupes (×2.5) and the cracked teeth were disregarded from the study. Moreover, considering WHO guidelines, the study samples were confirmed to be caries free [21]. Ultimately, fifteen polished enamel slices were selected for the rest of the study.

Demineralization
In order to induce an extremely rough surface, the polished enamels were etched with 35% phosphoric acid (Ultra-Etch, Ultradent products, USA) for 20 seconds and then washed for another 20 seconds with water spray.

Atomic force microscopy (AFM)
After erosive process, the surface topography AFM images of the sample were analyzed by Scanning Probe Briefly, three regions (5m5m) were randomly chosen on each flat enamel surface.

Remineralizing treatment
The fifteen samples were randomly divided into three groups (n=5) and were exposed to the following agents: (1) the experimental sesame gel, (2) 2% sodium fluoride gel (Masterdent, USA), which was served as positive control, and (3) distilled water (as negative control).
The samples in each group received 0.5cc of either treatment agents for 3minutes at 0, 8, 24, and 48h and washed by distilled water after each cycle. The specimens were stored in distilled water between these intervals [22].
The samples were then washed thoroughly by distilled water for 3 minutes to ensure the remnants of the agents were removed. Finally, the surface roughness of each enamel slice was measured again in three regions by the AFM.

Statistical analysis
The roughness values measured before and after remineralizing treatments in each group were compared, using paired t-test. The values of the three groups, yielded before or after treatment, were compared with each other by employing one-way ANOVA followed by Tukey post Hoc tests (=0.05).

Results
The sample AFM micrographs are demonstrated in    (Table 1).

Discussion
Our results showed that applying either fluoride gel or This finding is in accordance with our previous investigation, which reported enhanced surface microhardness of enamel exposed to a sesame-extracted gel [21]. Since the process of preparing the sesame gel was completely similar in both studies, it could be inferred that the relative smoother surface of the enamel in sesame group in the current study might be the result of elevated superficial mineral content. In this regard, we showed that the application of sesame gel on the tooth surface increases the microhardness of the enamel, but we  [21]. In that study, sesame extract was in gel form to facilitate its placement on the tooth surface. However, we did not investigate the remineralizing effect of sesame nor its impact on surface irregularities. Since reducing the surface irregularities might diminish the plaque accumulation on the surface enamel, it may prevent caries initiation.
Sesame seeds are rich in oil (50%) and protein (20%) [23]. The study of Kamchan et al. [14] reported that sesame seed has a high level of calcium but has limited bioavailability because of the presence of oxalate, phytate and fiber [14]. Poneros-Schneier et al. [24] investigated the bioavailability of calcium in several foods were investigated, and reported that sesame oil had a calcium bioavailability of about 65% [24]. In addition, some studies claimed about the drastic benefits of sesame for inhibition of plaque formation in clinical situations. Saravanan et al. [18] showed the effect of sesame oil on plaque-induced gingivitis, and found that sesame oil could reduce plaque development, gingivitis, and microbial count in oral cavity [18]. This effect can be due to not only the antimicrobial activity of sesame oil but also the cleansing effect of mouthwash.
Sesame is considered as a natural gift of calcium source [21]. Our study showed that for remineralization of etched enamel, the sesame extract was as effective as fluoride. This could be considered as an imperative finding because fluoride varnish is a synthetic product and its remineralizing capacity has been broadly proven.
Our natural sesame extract was statistically comparable to this kind of agent, which could be considered as a quite beneficial outcome in preventive dentistry. In other words, it seems that the sesame could be potentially considered as a natural gift for remineralization of the human tooth.
In this research, we turned the sesame extract into a gel to facilitate its placement on the tooth surface. However, the ease of access to that gel is debatable. In other words, if the evidence establishes that sesame oil has the same properties as its gel, it will be more reasonable to recommend sesame oil, since it is more easily available on the market.  [22,25].
In our study, the AFM micrograph showed the protective effect of sesame gel comparable to the fluoride gel. Since both types of these gels are water-soluble and the specimens were thoroughly washed with distilled water prior to AFM analysis, the reduced surface irregularities could not be attributed to the possible remained debris of the gels. Therefore, the observed surface smoothening would be the result of chemical deposition. Consequently, the sesame gel could be considered as re-hardening agent for enamel surface, confirmed by the microhardness test in previous research [21]. Nevertheless, further investigations using other tools such as SEM/EDAX is strongly suggested in future studies.
It should be mentioned that the in vivo situation is somehow different from the natural clinical situation since the saliva as well as the acquired pellicle would alter the impact of this gel [22]. Accordingly, future clinical surveys are needed to evaluate the clinical effect of sesame on tooth remineralization.

Conclusion
The results of the current research revealed that both the fluoride gel and the experimental sesame gel had noticeable beneficial effect for compensating the surface roughness of human enamel. Therefore, the sesame ex-tract could be considered as a potential agent in preventive dentistry while further clinical researches are strongly suggested.